A model system for the characterization of molecular recognition events in molecularly imprinted polymers (MIPs) is presented. The use of a biologically inspired, three-point hydrogen-bonding motif and a thin film polymeric matrix allows for pre- and post-polymerization binding properties to be characterized by infrared spectroscopy. A method to determine binding constants was developed and utilized before and after cross-linking. These values showed a 10-fold decrease in binding after polymerization, which was attributed to an increase in molecular confinement after polymerization and a change in the local structural environment of the binding cavity. Transport of the guest molecule was shown to be reversible.